In collaboration with Dr. John Bohnsack at the University of Utah, we studied the population structure of group B streptococci (GBS) isolated from infected and colonized neonates collected from the NICHD multi center GBS study in six U.S. academic centers from 1995 to 1999. The phylogenetic lineage of each GBS isolate was determined by multi-locus sequence typing, and each isolate was assigned a sequence type (ST). Isolates were clustered into clonal complexes (CC). A total of 899 neonatal GBS isolates were studied, of which 129 were associated with invasive disease. Almost all invasive and colonizing serotypes Ia, Ib, and V isolates were from single clonal complexes: 353/383 (92%) type I were CC 23, 78/81 (96%) type Ib were CC 12, and 134/153 (88%) type V were CC 1, respectively. In contrast, serotype II and III strains were mostly from two clonal complexes, CC 17 and CC 19. CC 17 serotype II GBS were isolated from a higher percentage of infected infants than from colonized infants, a result similar to that previously described for CC 17 serotype III GBS. While the vast majority of invasive GBS were confined to the same lineages that colonized neonates, there were also 9 invasive GBS isolates from lineages that were uncommon among colonizing isolates.